Inhibition of Src phosphorylation reduces damage to the blood-brain barrier following transient focal cerebral ischemia in rats

Bai,Yongsheng; Xu,Guanghui; Xu,Mengxue; Li,Qi; Qin,Xinyue

doi:10.3892/ijmm.2014.1946

Inhibition of Src phosphorylation reduces damage to the blood-brain barrier following transient focal cerebral ischemia in rats

Authors:
- Yongsheng Bai
- Guanghui Xu
- Mengxue Xu
- Qi Li
- Xinyue Qin
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Affiliations: Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China
Published online on: September 24, 2014 https://doi.org/10.3892/ijmm.2014.1946
Pages: 1473-1482
Copyright: © Bai et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

The disruption of the blood-brain barrier (BBB) caused by cerebral ischemia determines the extent of injury and patient prognosis. Inhibitors of Src can markedly minimize the infarct size and preserve neurological function. The Src protein tyrosine kinase (PTK) inhibitor, PP2, protects the rat brain against ischemic injury, possibly through the reduction of vascular endothelial growth factor A (VEGFA) expression and the upregulation of claudin-5 expression, which preserves the integrity of the BBB. In this study, the expression levels of phosphorylated (p)-Src, VEGFA and claudin-5 were determined to investigate the changes occurring in the levels of these proteins and to determine the benefits of PP2 treatment following cerebral ischemia/reperfusion (I/R). Our study included a sham-operated group, an I/R group, a vehicle-treated group (V) and a PP2-treated group (PP2). We found that the rats in the PP2 group exhibited greater preservation of neurological function and reduced VEGFA and p-Src protein expression compared with the rats in the I/R and V groups. Moreover, the mRNA and protein levels of claudin-5 were markedly higher in the PP2 group than in the I/R group or the V group after 3 days of reperfusion. Immunofluorescence staining revealed that the co-localized immunostaining of fibrinogen and claudin-5 was reduced in the PP2 group, which suggests that the exudation of fibrinogen in this group was less than that in the I/R and V groups. Furthermore, the reduced co-localization of immunostaining of glial fibrillary acidic protein (GFAP) and claudin-5 indicated that the rats in the PP2 group had only a slight disruption of the BBB. These findings suggested that PP2 treatment attenuated the disruption of the BBB following ischemia and minimized the neurological deficit; these effects were associated with a decreased VEGFA expression and an increased claudin-5 expression. Members of the Src PTK family may be critical targets for the protection of the BBB following cerebral ischemia.

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1	Ayata C and Ropper AH: Ischaemic brain oedema. J Clin Neurosci. 9:113–124. 2002. View Article : Google Scholar
2	Hatashita S and Hoff JT: Role of blood-brain barrier permeability in focal ischemic brain edema. Adv Neurol. 52:327–333. 1990.PubMed/NCBI
3	Ames A III, Wright RL, Kowada M, Thurston JM and Majno G: Cerebral ischemia. II. The no-reflow phenomenon. Am J Pathol. 52:437–453. 1968.PubMed/NCBI
4	Dvorak HF, Brown LF, Detmar M and Dvorak AM: Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol. 146:1029–1039. 1995.PubMed/NCBI
5	Dobrogowska DH, Lossinsky AS, Tarnawski M and Vorbrodt AW: Increased blood-brain barrier permeability and endothelial abnormalities induced by vascular endothelial growth factor. J Neurocytol. 27:163–173. 1998. View Article : Google Scholar : PubMed/NCBI
6	Kovács Z, Ikezaki K, Samoto K, Inamura T and Fukui M: VEGF and flt. Expression time kinetics in rat brain infarct. Stroke. 27:1865–1873. 1996.PubMed/NCBI
7	Hayashi T, Abe K, Suzuki H and Itoyama Y: Rapid induction of vascular endothelial growth factor gene expression after transient middle cerebral artery occlusion in rats. Stroke. 28:2039–2044. 1997. View Article : Google Scholar : PubMed/NCBI
8	Lennmyr F, Ata KA, Funa K, Olsson Y and Terént A: Expression of vascular endothelial growth factor (VEGF) and its receptors (Flt-1 and Flk-1) following permanent and transient occlusion of the middle cerebral artery in the rat. J Neuropathol Exp Neurol. 57:874–882. 1998. View Article : Google Scholar : PubMed/NCBI
9	Zhang ZG, Zhang L, Tsang W, et al: Correlation of VEGF and angiopoietin expression with disruption of blood-brain barrier and angiogenesis after focal cerebral ischemia. J Cereb Blood Flow Metab. 22:379–392. 2002. View Article : Google Scholar : PubMed/NCBI
10	Zhang ZG, Zhang L, Jiang Q, et al: VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest. 106:829–838. 2000. View Article : Google Scholar : PubMed/NCBI
11	Levy DE and Darnell J: Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 3:829–838. 2002.
12	Turkson J: STAT proteins as novel targets for cancer drug discovery. Exp Opin Ther Targets. 8:409–422. 2004. View Article : Google Scholar : PubMed/NCBI
13	Paul R, Zhang ZG, Eliceiri BP, et al: Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke. Nat Med. 7:222–227. 2001. View Article : Google Scholar : PubMed/NCBI
14	Niu G, Wright KL, Huang M, et al: Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene. 21:2000–2008. 2002. View Article : Google Scholar : PubMed/NCBI
15	Anderson JM: Molecular structure of tight junctions and their role in epithelial transport. News Physiol Sci. 16:126–130. 2001.PubMed/NCBI
16	Argaw AT, Gurfein BT, Zhang Y, Zameer A and John GR: VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown. Proc Natl Acad Sci USA. 106:1977–1982. 2009. View Article : Google Scholar : PubMed/NCBI
17	Longa EZ, Weinstein PR, Carlson S and Cummins R: Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 20:84–91. 1989. View Article : Google Scholar : PubMed/NCBI
18	Liu DZ, Ander BP, Xu H, et al: Blood-brain barrier breakdown and repair by Src after thrombin-induced injury. Ann Neurol. 67:526–533. 2010. View Article : Google Scholar : PubMed/NCBI
19	Chen J, Li Y, Wang L, et al: Therapeutic benefit of intravenous administration of bone marrow stromal cells (MSCs) after cerebral ischemia in rats. Stroke. 32:1005–1011. 2001. View Article : Google Scholar : PubMed/NCBI
20	Campbell M, Hanrahan F, Gobbo OL, et al: Targeted suppression of claudin-5 decreases cerebral oedema and improves cognitive outcome following traumatic brain injury. Nat Commun. 3:8492012. View Article : Google Scholar : PubMed/NCBI
21	Zlokovic BV: The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron. 57:178–201. 2008. View Article : Google Scholar : PubMed/NCBI
22	Rosenberg GA and Yang Y: Vasogenic edema due to tight junction disruption by matrix metalloproteinases in cerebral ischemia. Neurosurg Focus. 22:E42007. View Article : Google Scholar : PubMed/NCBI
23	Shichita T, Sugiyama Y, Ooboshi H, et al: Pivotal role of cerebral interleukin-17-producing gammadeltaT cells in the delayed phase of ischemic brain injury. Nat Med. 15:946–950. 2009. View Article : Google Scholar : PubMed/NCBI
24	Johnson FM, Saigal B, Talpaz M and Donato NJ: Dasatinib (BMS-354825) tyrosine kinase inhibitor suppresses invasion and induces cell cycle arrest and apoptosis of head and neck squamous cell carcinoma and non-small cell lung cancer cells. Clin Cancer Res. 11:6924–6932. 2005. View Article : Google Scholar : PubMed/NCBI
25	Desai CJ, Sun Q and Zinn K: Tyrosine phosphorylation and axon guidance: of mice and flies. Curr Opin Neurobiol. 7:70–74. 1997. View Article : Google Scholar : PubMed/NCBI
26	Lennmyr F, Ericsson A, Gerwins P, Akterin S, Ahlström H and Terént A: Src family kinase-inhibitor PP2 reduces focal ischemic brain injury. Acta Neurol Scand. 110:175–179. 2004. View Article : Google Scholar : PubMed/NCBI
27	Takenaga Y, Takagi N, Murotomi K, Tanonaka K and Takeo S: Inhibition of Src activity decreases tyrosine phosphorylation of occludin in brain capillaries and attenuates increase in permeability of the blood-brain barrier after transient focal cerebral ischemia. J Cereb Blood Flow Metab. 29:1099–1108. 2009. View Article : Google Scholar
28	Korematsu K, Goto S, Nagahiro S and Ushio Y: Microglial response to transient focal cerebral ischemia: an immunocytochemical study on the rat cerebral cortex using anti-phosphotyrosine antibody. J Cereb Blood Flow Metab. 14:825–830. 1994. View Article : Google Scholar : PubMed/NCBI
29	Okutani D, Lodyga M, Han B and Liu M: Src protein tyrosine kinase family and acute inflammatory responses. Am J Physiol Lung Cell Mol Physiol. 291:L129–L141. 2006. View Article : Google Scholar : PubMed/NCBI
30	Mukhopadhyay D, Tsokias L, Zhou X, Foster D, Brugge J and Sukhatme V: Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature. 375:577–581. 1995. View Article : Google Scholar : PubMed/NCBI
31	Bjorge JD, Jakymiw A and Fujita DJ: Selected glimpses into the activation and function of Src kinase. Oncogene. 19:5620–5635. 2000. View Article : Google Scholar : PubMed/NCBI
32	Zan L, Zhang X, Xi Y, Wu H, Song Y, Teng G, Li H, Qi J and Wang J: Src regulates angiogenic factors and vascular permeability after focal cerebral ischemia-reperfusion. Neuroscience. 262:118–128. 2014. View Article : Google Scholar : PubMed/NCBI
33	Morita K, Sasaki H, Furuse M and Tsukita S: Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. Journal Cell Biol. 147:185–194. 1999. View Article : Google Scholar : PubMed/NCBI
34	Huang ZG, Xue D, Karbalai H, et al: Biphasic opening of the blood-brain barrier following transient focal ischemia: effects of hypothermia. Canad J Neurol Sci. 26:298–304. 1999. View Article : Google Scholar : PubMed/NCBI
35	Jiao H, Wang Z, Liu Y, Wang P and Xue Y: Specific role of tight junction proteins claudin-5, occludin, and ZO-1 of the blood-brain barrier in a focal cerebral ischemic insult. J Mol Neurosci. 44:130–139. 2011. View Article : Google Scholar : PubMed/NCBI
36	Liu J, Jin X, Liu KJ and Liu W: Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood-brain barrier damage in early ischemic stroke stage. J Neurosci. 32:3044–3057. 2012. View Article : Google Scholar
37	Nag S, Venugopalan R and Stewart DJ: Increased caveolin-1 expression precedes decreased expression of occludin and claudin-5 during blood-brain barrier breakdown. Acta Neuropathol. 114:459–469. 2007. View Article : Google Scholar : PubMed/NCBI
38	Brown H, Hien T, Day N, et al: Evidence of blood-brain barrier dysfunction in human cerebral malaria. Neuropathol Appl Neurobiol. 25:331–340. 1999. View Article : Google Scholar : PubMed/NCBI
39	Kirk J, Plumb J, Mirakhur M and McQuaid S: Tight junctional abnormality in multiple sclerosis white matter affects all calibres of vessel and is associated with blood-brain barrier leakage and active demyelination. J Pathol. 201:319–327. 2003. View Article : Google Scholar : PubMed/NCBI
40	Tsukita S, Furuse M and Itoh M: Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol. 2:285–293. 2001. View Article : Google Scholar : PubMed/NCBI
41	Lo EH, Dalkara T and Moskowitz MA: Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci. 4:399–415. 2003. View Article : Google Scholar : PubMed/NCBI
42	Huang N, Ashrafpour H, Levine RH, Forrest CR, Neligan PC, Lipa JE and Pang CY: Vasorelaxation effect and mechanism of action of vascular endothelial growth factor-165 in isolated perfused human skin flaps. J Surg Res. 172:177–186. 2012. View Article : Google Scholar : PubMed/NCBI
43	Nitta T, Hata M, Gotoh S, et al: Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol. 161:653–660. 2003. View Article : Google Scholar : PubMed/NCBI
44	Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, Takano H, Noda T and Tsukita S: Complex phenotype of mice lacking occludin, a component of tight junction strands. Mol Biol Cell. 11:4131–4142. 2000. View Article : Google Scholar : PubMed/NCBI